Investigation of arcobacters in meat and faecal samples of clinically healthy cattle in Turkey

AIMS: To investigate the presence of Arcobacter spp. in minced beef meat (n = 97) and rectal faecal samples (n = 200) collected from cattle immediately after slaughter at a local abattoir in Turkey. METHODS AND RESULTS: Meat samples were examined using three different isolation procedures (CAT-supplemented media, de Boer arcobacter isolation method and membrane filtration method), but only one method (CAT-supplemented media) was employed for faecal samples. The isolated Arcobacter strains were identified by genus- and species-(multiplex) specific PCR assays. Arcobacter spp. were isolated from 5 and 9.5% of meat and faecal samples respectively. Although the only Arcobacter sp. found in meat samples was Arcobacter butzleri, all three pathogenic species--A. butzleri, A. cryaerophilus and A. skirrowii--were detected in the rectal swabs. No Arcobacter was isolated when the de Boer method was used for minced meat samples but the same five meat samples were found positive for arcobacters when CAT-supplemented media and membrane filtration method were used. CONCLUSIONS: The membrane filtration method was found to be superior to the CAT-supplemented media, because it led to a reduction in competing microflora. However, the necessity for one filter and medium for each sample makes this method somewhat expensive. The multiplex-PCR (m-PCR) assay shortened significantly the time required for the identification of Arcobacter spp. and also removed the possibility of false positive results due to other campylobacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the isolation of Arcobacter spp. in cattle for the first time in Turkey. The m-PCR assay enables the identification and differentiation of all arcobacters simultaneously in one-step PCR.     

Pet cats as carriers of Arcobacter spp. in Southern Italy

Aims:  To evaluate the presence of Arcobacter spp. in different biological samples from domestic cats in Southern Italy by using a species-specific PCR assay and thus to elucidate their potential significance as sources of human infection.
Methods and Results:  We investigated the prevalence of Arcobacter DNA in oral swabs, in peripheral blood samples and fine needle lymph node aspirate specimens from 85 cats of which 17 were clinically healthy and 68 had clinical signs of oral disease or lymphadenomegaly. Overall, molecular analysis has shown that Arcobacter -specific DNA was found in 78·8% (67 of 85) of all the cats. In the 67 Arcobacter -positive cats, 66 (77·6%) and 29 (34·1%) were found positive for Arcobacter butzleri and Arcobacter cryaerophilus, respectively. None of the examined samples gave a PCR product for Arcobacter skirrowii .
Conclusions:  This study demonstrates that pet cats commonly carry Arcobacter in the oral cavity. According to the clinical data, the Arcobacter detection results showed no significant difference between cats with oral pathology and those suffering from other different pathologies.
Significance and Impact of the Study:  Pet cats harbour Arcobacter spp. and may play a role in their dissemination in the domestic habitat. The high prevalence in a limited number of cat samples in this study may be of significance.     

Arcobacter contamination on pre- and post-chilled bovine carcasses and in minced beef at retail

Aims:  The present study aimed to assess the Arcobacter contamination on bovine carcasses postevisceration and postcooling in two slaughterhouses and in ready-to-eat minced beef.
Methods and Results:  Carcasses ( n  = 247) were sampled at four sites in two slaughterhouses and 100 minced beef samples were collected at retail. Isolation was performed by a quantitative and qualitative Arcobacter selective method, and the isolates were identified by multiplex PCR, after which a part of them were characterized by enterobacterial repetitive intergenic consensus (ERIC)-PCR. Although arcobacters were isolated from 37% of the bovine carcasses postevisceration with the chest and the foreleg as most contaminated sites, cooling the carcasses for at least 24 h reduced the incidence of Arcobacter (7%) on the carcass surface significantly. Arcobacter butzleri was the species most frequently isolated, although co-contamination with multiple species also occurred. At retail, arcobacters were present in 9% of the minced beef samples, with Arcobacter butzleri as the dominant species.
Conclusions:  Forced air cooling of bovine carcasses for at least 24 h decreased the number of positive carcasses, but did not eliminate all arcobacters.
Significance and Impact of the study:  This study demonstrates that maintaining good hygiene practices throughout the food supply chain is crucial to ensure safe food products at the consumer level.     

Detection of Arcobacter spp. in the coastal environment of the Mediterranean Sea

The occurrence of Arcobacter spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in cultures and environmental samples. Only one of the Arcobacter species, A. butzleri, was isolated from seawater and plankton samples. With some samples the A. butzleri PCR assay gave amplified products when cultures were negative. A. cryaerophilus and A. skirrowii were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of Arcobacter are present in marine environments. The assay was useful for detecting Arcobacter spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of A. butzleri and the presence of nonculturable Arcobacter spp. in the coastal environment of the Mediterranean Sea.     

Isolation and characterization of the emerging foodborn pathogen Arcobacter from human stool

At present, isolation of arcobacters from human specimens is performed by slightly of not modified Campylobacter, Yersinia or Leptospira isolation techniques, and knowledge if arcobacters are part of the human commensal flora is lacking. Therefore, an Arcobacter selective isolation procedure was validated for the examination of human fecal specimens, and the presence and characteristics of Arcobacter in feces of asymptomatic humans was examined in order to assess the clinical relevance of arcobacters in diarrheal stool. With this method, Arcobacter was isolated from seven of 500 (1.4%) stool samples of healthy people with Arcobacter cryaerophilus as the only species present. Seven A. cryaerophilus genotypes were detected and only one genotype was found per person. Neither A. butzleri nor A. skirrowii were isolated, therefore the presence of those latter species in clinical samples requires further attention. Though the pathogenic role and potential virulence factors of arcobacters have to be further examined, the current status of arcobacters as emerging pathogens remains justified.     

Prevalence and distribution of Arcobacter species in various sources in Turkey and molecular analysis of isolated strains by ERIC-PCR

AIMS: To determine the prevalence of Arcobacter in various food, animal and water sources in Turkey and to subtype the isolated strains using enterobacterial repetitive intergenic consensus (ERIC)-PCR. METHODS AND RESULTS: A total of 806 samples consisting of chicken (100) and turkey meat (100); minced beef (27); rectal swabs from cattle (173), sheep (68) and dogs (62); cloacal swabs of broilers (100) and layers (100); gall bladders of cattle (50) and drinking water samples (26) were examined. A previously described membrane filtration method was used for the isolation. Isolates were identified at species level using multiplex-PCR and discriminated by ERIC-PCR for subtyping. Ninety-eight (12.1%) of the samples examined were found positive for arcobacters. Arcobacter spp. were isolated from 68%, 4%, 6.9%, 8% and 37% of chicken and turkey meats, rectal swabs and gall bladders of cattle and minced beef, respectively. No arcobacters were obtained from the rectal swabs of sheep and dogs, cloacal swabs of broilers and layers, and water samples examined. In total, 99 Arcobacter isolates were obtained. Of these isolates, 92 were identified as Arcobacter butzleri, five were Arcobacter skirrowii and two were Arcobacter cryaerophilus. Thirteen distinct DNA profiles among A. butzleri isolates were obtained by the ERIC-PCR. Of these profiles, eight were from chicken carcass, three from cattle rectal swab and two from minced beef meat isolates. Some of the isolates originated from different sources gave the same DNA profiles. All isolates of A. skirrowii and A. cryaerophilus gave different DNA profiles. CONCLUSIONS: Poultry carcasses, minced beef meat, rectal swabs and gall bladders of cattle were found to be positive for Arcobacter spp. A. butzleri was the predominant species isolated. In addition, large heterogeneity among the Arcobacter isolates was determined. SIGNIFICANCE AND IMPACT OF THE STUDY: Contamination of the poultry carcasses and minced beef meat, rectal and gall bladder samples of cattle with arcobacters poses a risk for both human and animal infections. Detection of several different Arcobacter strains may suggest multiple sources for contamination and infection.     

Use of Hugh and Leifson's medium as a simple screening test to aid in the differentiation of Arcobacter spp. from background flora during their isolation from foodstuffs

Aims:  To investigate the suitability of Hugh and Leifson's medium (HLM) as the basis of a simple screening test to differentiate between contaminants and Arcobacter spp. during their isolation from foodstuffs.
Methods and Results:  Characterized Arcobacter spp. were obtained from recognized culture collections. Wild-type isolates of Arcobacter spp. and contaminants were obtained using published isolation protocols. Retail packs of red meats were used as the source of the isolates. Eighteen defined Arcobacter spp. gave no reaction on HLM, as did 10 local wild-type isolates. Overall 163 contaminants were studied for oxidative reactions on HLM and 86% of isolates demonstrated this property.
Conclusions:  HLM can usefully serve as a simple and effective screening test to differentiate between Arcobacter spp. and contaminants.
Significance and Impact of the Study:  Arcobacter isolation procedures are still being developed, and no effective diagnostic media currently exist. Rapidly excluding most contaminants can markedly increase the efficiency of isolation procedures by removing the need for extensive biotyping or the requirement to isolate DNA and conduct PCR tests.     

Development of a multiplex and real time PCR assay for the specific detection of Arcobacter butzleri and Arcobacter cryaerophilus

A new multiplex PCR and two specific TaqMan assays were developed to target the emerging pathogens A. butzleri and A. cryaerophilus. The assays also included an internal control to verify the presence of bacterial target DNA and amplification integrity. The multiplex assay used a published primer set (CRY1 and CRY2) for detecting A. cryaerophilus DNA (Houf, K., Tutenel, A., De Zutter, L., Van Hoof, J. and Vandamme, P., 2000. Development of a multiplex PCR assay for the simultaneous detection and identification of Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii. FEMS microbiology letters, 193 (1): 89-94.) and a novel A. butzleri primer set designed to target the rpoB/C gene sequences. To improve sample throughput and assay sensitivity a TaqMan assay for each Arcobacter spp. was developed which again utilised the heterogeneity contained in the rpoB/C and 23s rRNA gene sequences. The two TaqMan assays provided >2 log improvement in detection sensitivity for both Arcobacter spp. compared with the multiplex PCR assay and were able to detect <10 CFU per PCR reaction. To evaluate the effectiveness of the Arcobacter TaqMan assays with field isolates the assays were used to screen DNA samples prepared from faecal, hide and environmental samples obtained from two meat processing plants. In these studies, the TaqMan assays revealed that 2/150 (1.3%) samples were A. butzleri-positive, 11/150 (7.3%) were A. cryaerophilus-positive and the identity of generated amplicons was confirmed by DNA sequencing. Our results show that these TaqMan assays provide improvements in sensitivity and species-representation over other published Arcobacter PCR assays and they are compatible with detecting Arcobacters in sub-optimal matrices.     

Diversity and prevalence of Arcobacter spp. in broiler chickens

Ninety-nine strains of Arcobacter spp., isolated from 10 chicken carcasses purchased from a supermarket and 15 chicken carcasses collected from a poultry abattoir, were speciated using a variety of phenotypic identification methods. All were tested using API Campy test strips and the 16-test (Preston) identification scheme developed for campylobacters. Fifty strains were selected for examination using a more comprehensive probabilistic identification scheme, and the identity of representative strains confirmed by protein profiling using SDS-PAGE. All 25 carcasses yielded Arcobacter butzleri . Three supermarket and 10 abattoir carcasses also carried A. cryaerophilus , and two abattoir carcasses carried A. skirrowii . The API Campy scheme proved unsatisfactory for identifying these strains: only 20 of 99 strains were accurately identified, all of which were A. cryaerophilus , the only Arcobacter sp. included in the database. Moreover, 76 of 99 strains were misidentified. The 16-test scheme identified all the arcobacter strains as A. cryaerophilus, since neither A. butzleri nor A. skirrowii had been described when the scheme was developed. The computer-assisted probabilistic scheme succeeded in identifying all but one strain, the identity of which was clarified by the use of SDS-PAGE. To our knowledge this is the first time that arcobacters other than A. butzleri have been reported in poultry meat or any other food of animal origin. Their high prevalence in poultry products may be of significance to public health.     

Detection and enumeration of Arcobacter spp. in the coastal environment of the Straits of Messina (Italy)

Seawater and plankton samples from the Straits of Messina, Italy, were analysed to confirm the occurrence of potentially pathogenic Arcobacter butzleri, A. cryaerophilus and A. skirrowii. Both classical cultural methods and molecular techniques were used as confirmative steps of the growth in enrichment broth to enumerate and differentiate these bacteria. Only A. butzleri was isolated from seawater and plankton samples and was more abundant when associated with plankton than free-living. A. cryaerophilus was occasionally detected by PCR assay from environmental samples. The PCR procedure, used in a combined method, was useful in enumerating Arcobacter spp. in marine environment.     

Prevalence of thermotolerant Campylobacter in partridges (Perdix perdix)

Aim:  To estimate the prevalence of thermotolerant Campylobacter spp. in commercially reared partridges ( Perdix perdix ) in southern Italy.
Methods and Results:  Cloacal swabs of partridges ( n  =   240), equally distributed between male and female birds, from a game bird farm located in the Southern Italy were examined for the prevalence of thermotolerant Campylobacter spp. The samples were processed in order to detect thermotolerant Campylobacter spp. by culture methods. The positive samples were then confirmed by multiplex polymerase chain reaction. Thermotolerant Campylobacter spp. were isolated from 118 (49·2%) of the 240 cloacal swabs examined. As proved by PCR, 100% of the strains were identified as Campylobacter coli (118/118), and 15 (12·7%) out of the 118 positive samples were also positive for Campylobacter jejuni . In contrast, Campylobacter lari was not identified. Adult partridges showed a significantly higher prevalence ( P  <   0·05) than younger ones.
Conclusion:  These results reinforce the assumption that game birds may be considered as potential carriers of Campylobacter spp. for human being and other animal species.
Significance and Impact of the Study:  Although an earlier 1986 publication described the prevalence of Campylobacter coli in commercially reared partridges, this is the first report to confirm the species of Campylobacter using a PCR test.     

Ecology of Arcobacter species in chicken rearing and processing

AIMS: To investigate whether Arcobacter spp. colonize the poultry-rearing environment or whether they are contaminants acquired during transportation and/or from the processing plant. METHODS AND RESULTS: Samples were collected on poultry farms and in the processing plant during slaughter and dressing. Two cultural methods of detection were used. Isolates were identified to species level using a multiplex-polymerase chain reaction (m-PCR) method, either on the initial suspensions, or after enrichment, or on pure cultures of isolates. Of the 62 samples examined from poultry farms, arcobacters were found only outside the rearing sheds (in effluent sludge and stagnant water). Thirty-four samples were examined from the processing plant and 26 were positive for arcobacters. All the isolates were Arcobacter butzleri. Arcobacters were not found in any sample by direct plating nor by m-PCR on the initial suspensions, thus it was concluded that numbers were very low. CONCLUSIONS: Arcobacter spp. were not found in samples from the live birds and their immediate environment, but A. butzleri was found in effluent sludge and stagnant water outside the rearing sheds. However, A. butzleri is common in poultry abattoirs, and it appears that poultry carcasses are contaminated during processing. SIGNIFICANCE AND IMPACT OF THE STUDY: Arcobacters are not found inside poultry-rearing sheds, but are contaminants in the processing environment.     

Detection of Arcobacter spp. in piggery effluent and effluent-irrigated soils in southeast Queensland

AIMS: To investigate the occurrence and levels of Arcobacter spp. in pig effluent ponds and effluent-treated soil. METHODS AND RESULTS: A Most Probable Number (MPN) method was developed to assess the levels of Arcobacter spp. in seven pig effluent ponds and six effluent-treated soils, immediately after effluent irrigation. Arcobacter spp. levels in the effluent ponds varied from 6.5 x 10(5) to 1.1 x 10(8) MPN 100 ml(-1) and in freshly irrigated soils from 9.5 x 10(2) to 2.8 x 10(4) MPN g(-1) in all piggery environments tested. Eighty-three Arcobacter isolates were subjected to an abbreviated phenotypic test scheme and examined using a multiplex polymerase chain reaction (PCR). The PCR identified 35% of these isolates as Arcobacter butzleri, 49% as Arcobacter cryaerophilus while 16% gave no band. All 13 nonreactive isolates were subjected to partial 16S rDNA sequencing and showed a high similarity (>99%) to Arcobacter cibarius. CONCLUSIONS: A. butzleri, A. cryaerophilus and A. cibarius were isolated from both piggery effluent and effluent-irrigated soil, at levels suggestive of good survival in the effluent pond. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide quantitative information on Arcobacter spp. levels in piggery effluent and to associate A. cibarius with pigs and piggery effluent environments.     

Presence of Arcobacter spp. in environmental waters correlates with high levels of fecal pollution

We investigated the presence of Arcobacter spp. in 205 water samples of freshwater, seawater and sewage in Spain. These bacteria were present in 55.1% of the samples (113/205) and were significantly associated for the first time with bacterial indicators of fecal pollution. The dominant species in the positive samples was Arcobacter butzleri (94%) followed by Arcobacter cryaerophilus (30%) and Arcobacter skirrowii (1.8%).     

Survival capacity in water of Arcobacter species under different temperature conditions

Aims:  To assess the survival capacity in vitro of arcobacters in water at temperatures applied in the food industry.
Methods and Results:  Four strains of each Arcobacter species were inoculated in potable water and water with 1% organic material and stored at 4, 7, 20, 52, 56 and 60°C. Samples were taken at known time points and the numbers of bacteria were determined on Arcobacter -selective medium. All Arcobacter species remained viable for a temperature-dependent period of time, although Arcobacter butzleri displayed a significant longer survival and heat resistance . No significant intraspecies differences were detected, resulting in no definite identification of origin or strain dependency. The survival period for all species was prolonged in the presence of the organic material only for the low temperatures.
Conclusions:  The present study demonstrates that water can act as a reservoir and as a potential source of Arcobacter contamination to humans and animals.
Significance and Impact of the Study:  This study assessed for the first time the survival of all human-related Arcobacter species in water. Particularly A. butzleri showed to be the most robust species with regard to temperature which is interesting as that species is often found in human clinical specimens.     

Differentiation of Arcobacter species by numerical analysis of AFLP profiles and description of a novel Arcobacter from pig abortions and turkey faeces

AIMS: To evaluate the efficacy of amplified fragment length polymorphism (AFLP)-based genetic profiling for taxonomic and epidemiological analyses of diverse Arcobacter species. METHODS AND RESULTS: Seventy-two isolates of A. butzleri, A. cryaerophilus, A. skirrowii and A. nitrofigilis, and a previously unclassified porcine abortion strain were studied. AFLP profiling was performed using a BglII-Csp6I-based protocol previously used to characterize Campylobacter species. Duplicate profiles of 20 isolates were 93.25% similar, indicating high reproducibility. Numerical analysis of all 72 strains revealed five phenons at the 29% similarity level, four of which represented each of the known species studied. The remaining phenon was further characterized by phenotypic and 16S rDNA sequence analyses, the results of which indicated it to be a novel Arcobacter species. The genetically distinct subgroups of A. cryaerophilus were differentiated at the 39.5% similarity level. For strain typing, 62 distinct types were defined, with evidence of clonal lineages within A. butzleri, A. cryaerophilus and A. skirrowii. CONCLUSIONS: AFLP profiling is an effective means of determining taxonomic and strain relationships for arcobacters. SIGNIFICANCE AND IMPACT OF THE STUDY: First use of AFLP profiling for diverse Arcobacter species; indication of clonality in A. butzleri, A. cryaerophilus and A. skirrowii; potentially novel Arcobacter taxon identified.     

Comparative study of a new quantitative real-time PCR targeting the xylulose-5-phosphate/fructose-6-phosphate phosphoketolase bifidobacterial gene (xfp) in faecal samples with two fluorescence in situ hybridization methods

Aims:  To detect and enumerate bifidobacteria in faeces with a new quantitative multiplex real-time PCR (qPCR) method and to compare the results obtained with fluorescence in situ hybridization (FISH) methods.
Methods and Results:  A multiplex qPCR assay was developed, which enabled the enumeration of Bifidobacterium spp. by targeting the bifidobacterial xylulose-5-phosphate/fructose-6-phosphate phosphoketolase gene ( xfp ) and total bacteria using universal Eub-primers targeting 16S rRNA gene from the domain bacteria. The qPCR assay showed high sensitivity and specificity and a low detection limit of about 2·5 × 10³ bifidobacterial cells per gram of faeces. The qPCR results were compared with FISH combined with microscopy or flow cytometry (FCM). No statistical differences among bifidobacterial counts averages measured in adult faeces with the three methods were observed. Total bacterial count averages were higher with the FISH method coupled with microscopic analyses compared to FISH with FCM, whereas total cell numbers estimated by qPCR were intermediate between the two FISH methods.
Conclusions:  The new qPCR assay was shown to be sensitive, rapid and accurate for enumerating bifidobacteria in faeces.
Significance and Impact of the Study:  This method is a valuable alternative for other molecular methods for detecting faecal bifidobacteria, especially when their counts are below the detection limit of the FISH methods.     

Development of a combined PCR-culture technique for the rapid detection of Arcobacter spp. in chicken meat

A combined PCR-culture technique was developed to detect Arcobacter spp. in fresh chicken meat. Following a short selective enrichment of chicken samples, bacterial DNA was extracted and amplified using primers targeted at the genes encoding 16S rRNA of Arcobacter spp. The selected primers amplify a 181-bp fragment from all Arcobacter spp., whereas no PCR product is generated for other bacteria, including the closely related Campylobacter and Helicobacter species. The assay was used to screen 96 retail-purchased chicken samples for the presence of Arcobacter spp. Fifty-three percent of the samples analysed were positive for this micro-organism. The assay is simple and sensitive and reduces the amount of time required to positively detect Arcobacter spp. in poultry meat.     

Arcobacter population dynamics in pigs on farrow-to-finish farms

Healthy pigs are an important reservoir for the emerging human pathogen Arcobacter which can result in contamination of porcine carcasses and pork and the spread of arcobacters into the environment. Up to now, the excretion of arcobacters by pigs has been studied, but information about the transmission routes in fattening pigs is lacking. The present study aimed to elucidate the Arcobacter population dynamics in pigs during the fattening period on four farrow-to-finish farms. On each farm, 30 clinically healthy, 12-week-old piglets were selected. Fecal samples were collected on 10 sampling occasions until a slaughter age of 30 weeks was reached. Arcobacter spp. were isolated by a selective method and identified by multiplex PCR. The genetic diversity was examined by amplified fragment length polymorphism and enterobacterial repetitive intergenic consensus PCR. The Arcobacter presence in the fecal samples on the four farms ranged from 11.3 to 50.0%, with excretion levels of up to 10(4) CFU/g feces. The ratio in which Arcobacter species were isolated varied between the farms and over time. Characterization revealed a high degree of genotypic diversity among the isolates. Arcobacter strains persisted and spread within the finishing unit during the fattening period. The occurrence of both unique and shared genotypes in pigs in adjacent and nonadjacent pens demonstrates that transmission routes other than fecal-oral transmission occur.     

Prevalence of campylobacters and arcobacters in ducks at the abattoir

Ten duck carcasses, five from each of two different flocks, and four pairs of pooled duck caecal contents, each pair from a separate flock, were examined by a variety of techniques for arcobacters and campylobacters. Campylobacter coli, C. jejuni ssp. jejuni , C. upsaliensis, Arcobacter cryaerophilus and A. butzleri were isolated from duck caecal contents. Campylobacter coli, C. jejuni ssp. jejuni, A. cryaerophilus, A. butzleri and A. skirrowii were isolated from carcasses. The most effective methods for isolating these bacteria from carcasses involved selective enrichment in campylobacter enrichment broth, containing a cefoperazone, amphotericin, teicoplanin supplement, followed by plating onto modified charcoal cefoperazone deoxycholate agar (mCCDA), or plating onto non-selective blood agar after filtration through a 0·65 μm pore size cellulose acetate filter. In contrast, recovery from caecal contents was most effective by direct plating onto mCCDA. API test strips performed poorly, failing to identify A. skirrowii or A. butzleri (which are not included in the scheme), or even many common campylobacters. The Preston biochemical characterization scheme was more helpful, though it did not distinguish between Arcobacter species. The species of most isolates of campylobacter, identified using the Preston scheme, was confirmed by the use of SDS-PAGE of whole cell proteins and this technique was also used successfully to speciate arcobacters.